The harvest of cells from tissue for maintenance and propagation in vitro by tissue culture is a major tool in medical and biochemical research. Tissue culture is the technique or process of propagating and/or supporting the metabolism of tissues or cells derived from organisms (plant or animal) in a formulated nutritive environment. Once isolated by gentle tissue dissociation, cells are incubated in nutritive media capable of supporting life functions. With few exceptions, cells require attachment to a substratum in order to perform normal metabolic functions, grow and divide. In tissue, the substratum which provides the support for cell growth is either the basement membrane or interstitial matrix which consists of collagen, laminin, fibronectin, etc. In vitro, this substratum is most often plastic, although glass and microporous cellulosic and other filters are sometimes used as substitutes. Examples of cell uses produced via tissue culture include: (1) the study of the metabolism of the cell, the effect of infectious agents (i.e., viruses, bacteria, etc.) on the cell, the interactive metabolism of different cell types (i.e., epithelial cells, fibroblasts, immuno-competent cells, thymocytes, platelets, etc.), the effect of exogenous factors on cellular metabolism, the genetic composition of cells (in vitro diagnostics); (2) the production of specific compounds, i.e., DNA, RNA, proteins or other cellular components; and (3) the re-implantation of cells as for skin, corneal grafts, brain, vascular grafts, and in vitro fertilization.
In recent years, collagen, laminin, fibronectin and other extracellular matrix components have been extracted and purified from animal tissues and marketed to cell and tissue culture researchers as cellular adhesion promoters. Synthetic poly-D-lysine and poly-L-lysine have also been sold for such purposes. The primary reason for this is that, in vitro, substrates such as plastic or glass are biologically inert and often do not provide sufficient substrate adhesion for adequate cell or tissue attachment. Specific examples illustrative of poor attachment efficiency include primary cell isolates, cells seeded at low densities, transfected cells, and cells seeded in continuous flow systems such as bio-reactors or hollow tube culture systems. In addition, certain substrates such as some microporous filters or Teflon.RTM. materials used for vascular grafts do not permit any cell attachment due to low surface energy.
Although cell adhesion promoters have assisted with attachment problems to a significant degree, certain inadequacies are still noteworthy. In particular, once attached to the substrate, most of these factors have a variable and inadequate shelf life.
It is, thus, one object of the present invention to provide coating compositions useful as cell adhesion promoters and stabilizers to facilitate or augment attachment efficiency, rate and/or strength of adhesion, growth and specialized function of cells to tissue culture or nontissue culture materials and substrates including plastic, glass, metals, microporous filters (cellulosic, nylon, glass fiber, polyester, polycarbonate, polyethylene terephthalate and other synthetic and nonsynthetic materials including other synthetic polymeric materials and products resulting from modifications made to the aforementioned synthetic polymeric materials), natural polymers or other nonsynthetic materials, and synthetic or alloplastic materials that may be used in tissue or prosthetic graft procedures (e.g., mechanical heart and polytetrafluoroethylene and related vascular grafting materials).
A second object of the present invention is to provide preparations useful as cell adhesion promoters to facilitate or augment attachment efficiency, rate and/or strength of adhesion of other biologically active moieties such as proteins, DNA, hormones and antibiotics to a variety of substrates, some of which are mentioned above.
Thus biologically active moieties such as proteins and other macromolecules can be coated onto substrate surfaces such as, for example, polystyrene surfaces, by noncovalent adsorption from aqueous solutions. The amount of these macromolecules absorbed onto such surfaces depends on the coating conditions (such as, for example, pH, temperature, ionic strength, ionic composition, concentration of reactants, etc.). The stability of the coating on the surface also depends on similar parameters, as well as the extent of the dryness of the coating and storage conditions (such as, for example, humidity, temperature, gaseous environment, etc.).
The inventors have found that the stability of poly-D-lysine coated surfaces strongly depends on the counter anion that is used during the coating process. Poly-D-lysine is very highly positively charged at neutral pH, and is therefore complexed with a counter anion on the coated surface. It has been found that by applying an improved coating composition of the present invention, such as, for example, a coating composition comprising poly-D-lysine in a citrate or sulfate solution (thus using citrate salt or sulfate salt as the counter anion), to various substrate surfaces, this will result in an increased shelf-life for these substrates. The results obtained are far superior to previous coating compositions which use chloride, acetate, EDTA, carbonate and PBS (phosphate/chloride combination) solution or water.
The present PDL BIOCOAT.RTM. Cellware product which is PDL in 1.times.PBS solution coated onto polystyrene surfaces, has a shelf-life that is dependent on the configuration of the substrate (whether it is a dish, flask or plate, as well as the size and number of cavities in the plates), storage temperature, humidity, packaging, etc. The shelf-life (i.e., stability) of a coated substrate can be enhanced at least two to three-fold by the method of the present invention.
The method of the present invention can be utilized to coat a variety of substrates, as described above and including, for example, plasticware, to increase the shelf-life of such products and allow room temperature storage for such products instead of requiring refrigeration at, for example, 4.degree. C. or lower temperatures. The method of the present invention results in a stability/increased shelf-life which is not dependent on the packaging of, for example, the plasticware.